Bovine vaccines and methods of making and using same

ABSTRACT

A polyvalent vaccine effective in immunization of bovines against infectious bovine rhinotracheitis virus (IBR), bovine viral diarrhea virus (BVD) and parainfluenza-3 virus (PI-3) is composed of a suspension, in a vehicle such as an aqueous solution of formaldehyde suitable for parenteral injection of the three viruses in killed form. The three viruses are separately propagated and subsequently suspended in aqueous solutions containing 0.4 percent by volume formalin, and the respective suspensions are maintained at a temperature of about 4*C. for 1 week. Portions, for example equal portions, of the three suspensions are combined to produce a polyvalent vaccine. An equal volume of an aqueous solution containing from 1 to 5 percent weight/volume of a soluble alginate, such as sodium alginate, having certain specified properties may be added to the vaccine as an adjuvant to enhance its effectiveness. Such polyvalent vaccines are adapted for parenteral administration in the vaccination of bovines.

United States Patent 1191 Shechmeister et al.

1 BOVINE VACCINES AND METHODS OF MAKING AND USING SAME [75] inventors:Isaac L. Shechmeister; Joseph R.

Kolar, Jr.; William G. Kammlade. Jr., all of Carbondale, 111.

173] Assignee: Southern Illinois University Foundation, Carbondale. 111.

22 Filed: Dec.27, 1973 [21] Appl. No.: 428,658

Related US. Application Data [62] Division of Set. No. 161.701, July 12.1971. abandoned. which is a division of Scr. No. 8.631. Feb. 4. 1970.Pat. No. 3.629.413.

[52] US. Cl. 424/89 [51] Int. Cl. A61K 27712 [58] Field of Search 424/89[56] References Cited UNITED STATES PATENTS 3.075.883 H1963 Scherr etal. 424/89 3.629.413 12/1971 Shechmeister et al 424/89 3.838.004 9/1974Mebus et a1. 424/89 OTHER PUBLICATIONS Hermodsson et al., Nature194(4831): 893-894, June 2, 1962, Properties of Bovine Virus DiarrheaVirus. Gillette, Diss. Abtr. 278: 363-364 (1966), CharacterizationStudies of Two Strains of Bovine Viral Diarrhea Virus."

Kolar et al., Am J. Vet. Res. 33(7): 1415-1420, July 1972 Use in Cattleof Formalin-killed Polyvalent Vaccine with Adjuvant against infectiousBovine Rhinotracheitis, Bovine Viral Diarrhea and Parainfluenza 3Viruses."

Shechmeister et al., Vet. Bull. 38, No. 1381 (1968). Abst. of Am. .1.Vet. Res. 28: 1373l378 (1967),"

1 Dec. 9, 1975 Use of Sodium Alginate Adjuvant in Immunization againstEquine influenza."

Femelius et al.. Am. J. Vet. Res. 32(12): 1963-1979. Dec. 1971.Evaluation of Soluble Antigen Vaccine Prepared from Bovine ViralDiarrhea Mucosal Disease Virus-infected Cell Cultures."

Femelius et al., Am .1. Vet. Res. 33(7): 142-1431 July 1972 Evaluationof B-Propiolactone-lnactivated and Chloroform Treated Virus VaccinesAgainst Bovine Viral Diarrhea Mucosal Disease."

Primary Examiner-Shep K. Rose Attorney. Agent. or Firm-Koenig.Senninger. Powers and Leavitt 57 ABSTRACT A polyvalent vaccine efiectivein immunization of bovines against infectious bovine rhinotracheitisvirus (1BR), bovine viral diarrhea virus (BVD) and parain fluenza-3virus (Pl-3) is composed of a suspension. in a vehicle such as anaqueous solution of formaldehyde suitable for parenteral injection ofthe three viruses in killed form. The three viruses are separatelypropagated and subsequently suspended in aqueous solutions containing0.4 percent by volume formalin. and the respective suspensions aremaintained at a temperature of about 4C. for 1 week. Portions, forexample equal portions, of the three suspensions are combined to producea polyvalent vaccine. An equal volume of an aqueous solution containingfrom 1 to 5 percent weight/volume of a soluble alginate, such as sodiumalginate, having certain specified properties may be added to thevaccine as an adjuvant to enhance its effectiveness. Such polyvalentvaccines are adapted for parenteral administration in the vaccination ofbovines.

3 Claims, 9 Drawing Figures US. Patent Dec. 9, 1975 Sheet 3 of93,925,544

cucfls oaooficocnsona 2. E 138, 3 3 3 g u n m x2 2. 8 8 8 5 .5? 8 2. 5m5? 83: 2:8 8 v8== i m M afivm Wyn;

MOE

US. Patent Dec. 9, 1975 Sheet 4 of9 3,925,544

co -Hmon on want a X 0..." IIIIS Jam uogdmspewa enema p rezxudpaa WE 5%?53 5 2 3 3 1 2 3 o m mm. 6 8 3 0 6 3 SS8 8 2. 5m 5 83: 2:3 3 u3i 4 US.Patent Dec. 9, 1975 Sheet 7 of9 3,925,544

:2 30?. ucoumm All apoFocwoaom oocm anaua 65; m Q n m nus .522 8:322. 5.z won-5 :oZmEEZam: mm ummmmaxo -Q m tan in. 6 *0 3459.00 a582, E2 3o0its ugfiauocm 23m. *0 02.232 anon-Z20 290m U.S. Patent Dec. 9, 1975Sheet 8 of9 3,925,544

c2353? 38 un can 2 cc am an E. 8

(lull n E 35m 3:3: *0 32:33. as vunnoaxu d m E in.

2; 3 woman-coo 2:02; 222 20;

:3! m-=02: 2:8 *0 omzoamoh 3252-5 @0293 mm m afiuaae go maoadpu QUECROSS-REFERENCE TO RELATED APPLICATION Feb. 4, 1970, now U.S. Pat. No.3,629,413 dated Dec. 21, 1971.

BACKGROUND OF THE INVENTION The present invention relates to vaccinesand more particularly to a polyvalent vaccine useful in the treatment ofinfectious diseases in bovines.

Three viruses which commonly infect bovines such as beef cattle areinfectious bovine rhinotracheitis (IRB), bovine viral diarrhea (BVD) andparainfluenza-3 (PI-3) also known as the shipping fever" virus. Becausethey effect beef cattle, these viruses have given rise to a commerciallysignificant problem. In the past, there have been available monovalentvaccines for the treatment of these respective viruses and a bivalentvaccine for the treatment of two of these three viruses. However, thesevaccines have been prepared from live viruses. The use of such vaccinescontaining live viruses has been contraindicated in the case of pregnantcows, and the effect of such vaccines on other animals has not beenfully evaluated. Also, there has not been available for treatment of theaforementioned viruses a vaccine containing an adjuvant to enhance itseffectiveness.

In summary, there has not heretofore been available a single polyvalentvaccine which is effective against the three aforementioned viruses,which can be recommeded and used in the treatment of all bovines andwhich requires only a single administration for effective protection ofsuch bovine animals instead of multiple administrations of severaldifferent vaccines.

SUMMARY OF THE INVENTION Among the objects of the invention may be notedthe provision of a novel polyvalent vaccine which is effective inimmunizing bovines against the three aforementioned viruses; theprovision of such a vaccine which can safely and reliably be used in thevaccination of all bovines; the provision of a polyvalent vaccine ofthis type which contains the respective viruses in killed form; theprovision of a vaccine of this character which produces an immuneresponse in bovines which lasts for an extended period of time; and theprovision of methods for preparing and using such polyvalent vaccines ina convenient and economical manner. Other objects and features will bein part apparent and in part pointed out hereinafter.

In accordance with the present invention, there is provided a polyvalentvaccine comprising a suspension, in a vehicle suitable for parenteralinjection, of killed infectious bovine rhinotracheitis virus, bovineviral diarrhea virus and parainfluenza-3 virus. The invention is alsodirected to the method of preparing such a polyvalent vaccine by firstseparately propagating the three viruses, suspending the respectiveviruses in separate aqueous solutions containing a final concentrationof about 0.4 percent by volume formalin to kill the viruses, maintainingeach suspension at a temperature of about 4C. for about 1 week, andcombining portions of each suspension to produce a polyvalent vaccinecontaining the three viruses in killed form. The invention furtherincludes the method of treating bovine infections by parenterallyadministering the novel polyvalent vaccines to bovines.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. I is a graph showing the averageantibody response of cattle to infectious bovine rhinotracheitis viruswhen inoculated with either infectious bovine rhinotracheitis monovalentvaccine or the polyvalent vaccine of the present invention;

FIG. 2 is a graph showing the average antibody response of cattle tobovine viral diarrhea virus when inoculated with either bovine viraldiarrhea monovalent vaccine or a polyvalent vaccine according to thepresent invention; a

FIG. 3 is a graph showing the average antibody of cattle toparainfluenza-3 virus when inoculated with either parainfluenza-3monovalent vaccine or a polyvalent vaccine according to the presentinvention;

FIG. 4 is a graph showing the average antibody of cattle toparainfluenza-3 virus when inoculated with either parainfluenza-3monovalent vaccine or a polyvalent vaccine in accordance with thepresent invention expressed on a different basis than in FIG. 3;

FIG. 5 is a graph showing the average antibody response of cattle toparainfluenza-3 virus when inoculated with either pairainfluenza-3monovalent vaccine or a polyvalent vaccine according to the presentinvention expressed on still another basis;

FIG. 6 is a graph showing the pooled antibody response of cattle toinfectious bovine rhinotracheitis virus when inoculated with apolyvalent vaccine of the present invention;

FIG. 7 is a graph showing the pooled antibody response of cattle tobovine viral diarrhea virus when inoculated with a polyvalent vaccine ofthe present invention FIG. 8 is a graph showing the average antibodyresponse of cattle to parainfluenza-3 virus when inoculated with apolyvalent vaccine of the present invention; and

FIG. 9 is a graph showing the pooled antibody response of cattle toparainfluenza-3 virus when inoculated with a polyvalent vaccine of thepresent invention expressed on a different basis than in FIG. 8.

DESCRIPTION OF THE PREFERRED EMBODIMENTS It has now been found, inaccordance with the present invention, that a polyvalent vaccinecontaining antigens of infectious bovine rhinotracheitis virus, bovineviral diarrhea virus and parainfluenza-3 virus, all in killed form, iseffective in the immunization of bovines against these viruses. Animalstudies, described in detail hereinafter, have demonstrated theeffectiveness of such a polyvalent vaccine against the infectiousdiseases caused by the viruses whose antigens are present in onevaccine.

The polyvalent vaccines of the ivention are readily prepared by firstseparately propagating the three aforementioned viruses as describedbelow. The three viruses are then separately suspended in an aqueousinactivating medium to kill the viruses. Preferably, the inactivatingmedium is an aqueous solution containing a final concentration of about0.4 percent by volume formalin. This final concentration may rangebetween about 0.3 percent and 0.5 percent by volume formalin. Formalinis a 37 percent by weight formaldehyde solution. The separatesuspensions are maintained at a temperature of about 4C. forapproximately I week. Portions of the three suspensions are thencombined to produce the complete polyvalent vaccine. Preferably, equalproportions of the three separate suspensions are combined to yield aneffective vaccine. However, these proportions may be varied somewhatwithout substantially affecting the results obtained.

Preferably, the final vaccine contains a concentration of between about10 and about organisms or units of each of the three killed viruses perml. of vaccine l0"-l0 TClD /mlJ. The dosages of vaccine employed incarrying out the invention may be varied widely, but preferably rangesbetween l and 10 ml., a dosage of 1 ml. being preferred when theconcentration of each of the killed viruses is about 10 and a dosage of10 ml. being preferred when the concentration of each of the killedviruses is about l0". It will be understood that the concentrations anddosages employed may both be varied within the above-noted ranges.

In order to enhance the effectiveness of such a polyvalent vaccine, asoluble alginate may be incorporated into the vaccine as an adjuvant.Such soluble alginates and their properties are fully disclosed inScherr et al. US. Pat. No. 3,075,883, dated Jan. 29, I963, incorporatedherein by reference. As disclosed therein, such soluble alginate may beutilized inthe form of an aqueous solution containing from I to 5percent weightvolume of a soluble alginate, such as sodium alginate,having the following characteristics:

a. in 5 percent weight/volume aqueous solution,

readily passes a 24-gauge needle;

b. in 4 percent weight/volume aqueous solution, has

a viscosity less than 50 centistokes; c. a chemical equivalent below250; and d. a milliosmolarity less than 150 per kg. of water on a Fiskeosmometer. Additionally, such an alginate solution may advantageouslycontain alginate insolubilizing ions in the form, for example, of aphysiologically acceptable gluconate such as calcium gluconate. Thevaccine should not, however, contain calcium or other insolubilizingions of a concentration sufficient to precipitate as the insolublealginate get before injection of the vaccine into animals is made.

In the practice of the present invention, we have found it satisfactoryto use a commercially available aqueous alginate solution of the abovetype which contains 4 percent weight/volume sodium alginate and 0.67percent by weight calcium gluconate. The calcium gluconate content mayrange between about 0.4 and 0.8 percent by weight. An equal volume, forexample, of such an aqueous solution may be added to the vaccine,prepared as described above, to further enhance its effectiveness and toproduce a higher level of antibodies.

The novel vaccines of the invention are prepared utilizing a vehicle,preferably an aqueous solution, suitable for parenteral injection as byintramuscular, intradermal or intracutaneous injection into bovineanimals.

As demonstrated by the animal studies described below, the vaccines ofthe invention produce a high level of antibodies in cattle inoculatedtherewith. Moreover, our vaccines, particularly those containing theabovedescribed adjuvant, produce an immune response which islong-lasting, i.e., for at least a period of 120 days.

Two studies were carries out to demonstrate the practice andeffectiveness of the vaccines and methods of the present invention.

In the first study, five vaccines were used. Two of these were complexpolyvalent vaccines. One contained a mixture of formalin-killedinfectious bovine rhinotracheitis (lBR), bovine viral diarrhea (BVD) andparainfiuenza-3 (Pl-3) viruses plus sodium alginate as an adjuvant. Theother vaccine contained a mixture of the same three killed viruseswithout the adjuvant. Three monovalent vaccines were also prepared,these containing sodium alginate as an adjuvantv and the killed lBR, BVDand Pl-3 viruses respectively. Each of the vaccines was suspended in asterile aqueous medium as a vehicle suitable for parenteraladministration.

All viruses used in preparing the above vaccines were grown in bovineembryonic trachea (BEmT) cell cultures. The cells were passed in minimumessential medium (MEM, of. Eagle, H., Amino Acid Metabolism in MammalianCell Cultures," Science, Vol. 130, p. 432-437, I959) with a fetal bovineserum concentration of l0 percent The cells for stocks and virusespropagation were serially passed in 16 02. glass prescription bottles. Asaline-trypsin-versene (STV) buffer solution was used to remove thecells from the surface of the glass. After centrifugation, the cellswere resuspended in MEM plus serum, distributed in 16 02. bottles at acencentration of6 X 10 cells per ml. (30 ml. per 16 oz. bottle), andincubated at 37C. A confluent cell monolayer formed in 4 days. Thebovine trachea cell line was used in the serum assay procedures and topropagate the viruses.

All viruses employed were propagated by inoculating 0.3 ml. of i0dilution stock virus into 16 oz. glass prescription bottles of washed,prepared bovine trachea cell monolayers and were incubated at 37C. untila desired titer was achieved (e.g. 4 days). After harvesting of thevirus, the tissue culture fluid containing the virus was tested forbacterial contamination by inoculation into thioglycollate medium andnutrient broth. The absence of bacterial growth after 3 days incubationat 37C. gave evidence that the virus cultures were free from bacteria.The viruses in the tissue culture fluid were then assayed forhamagglutinating activity and neutralization studies using themicrotiter technique.

Each of the viruses was suspended in an aqueous solution and inactivatedwith 0.4 percent formalin (final concentration) and maintained for 1week at 4C. The two polyvalent vaccines were prepared by combining equalproportions of the three killed viral components, i.e. the lBR, BVD andPl-3 killed viruses. The vaccines with adjuvants were prepared by mixingthe respective viral suspension or suspensions with a equal volume of anaqueous solution containing 4.0 percent weightlvolume sodium alginateand 0.67 percent by weight calcium gluconate. This soluble alginate hadthe characteristics previously set out. The vaccines were then testedfor bacterial contamination in nutrient and thioglycollate brothcultures and for residual viral activity in bovine embryonic trachea(BEmT) tissue culture.

The parainfluenza-3 (Pl-3), strain SF-4, of virus used for vaccineproduction was serially passed in BEmT cells and a hemagglutination (HA)titer of 1:512 was obtained. Infectious bovine rhinotracheitis (lBR) wasserially passed in the BEmT cells and a tissue culture inefective dose(TClD titer of 10 per ml. was obtained. The bovine viral diarrhea virus(BVD) was passed 15 times in BEmT cells and a TCID titer of IO -per ml.was achieved. The TCID titers were obtained by titerating the virus in asuspended cell system using a microtiter technique and thedeterminations were made using the method of Reed, L. J. and Muench, H.,A Simple Method of Estimating Fifty Percent Endpoints," Am. J. Hyg. 27,(1938): p.493-497.

Forty-five head of beef cattle, 23 heifers and 22 steers, of mixed Angusand Hereford breeds, each weighing 300-400 pounds, were employed as testanimals in the first study. The animals ranged in age from 5-8 months.They had no history of vaccination against any of PI-3, or IBR, or BVDviruses. All animals were in good health at the onset of vaccination andthere had been no history of clinical cases of shipping fever (PI-3)IBR, or BVD in the herd.

The 45 head of catter were divided into groups. Groups II and IIIconsisted of 3 and 2 animals, respectively, and the remaining eightgroups had five animals per group. Twenty-five ml. of blood was obtainedfrom each animal immediately prior to initial inoculation with vaccine.All serum samples were tested for antibodies against PI-3, IBR, and BVDusing the HI test or the neutralization test described infra. Thevaccines were parenterally administered by inoculating intramuscularlyin the biceps femoris with 10.0 ml. of vaccine or control mixture asindicated in Table 1. Cattle in Groups IV, V, VI, IX, X werereinoculated with an additional 10.0 ml. of an identical preparation 38days after the first injection. Blood samples of 25.0 ml. were obtainedfrom all cattle on the following days subsequent to the initialinjection: 3, 7, l0, I4, 17, 23, 31, 35, 38,45, 52,66, 87, and 120.Table I shows the composition of the vaccines used (prepared asdescribed above) and the injection protocol employed for the cattle inGroups l-X.

TABLE I FOR CA'I'I'LE IN GROUPS I-X erythrocytes are used. Guinea pigerythrocytes were used in the HA determinations.

The following dilutions of the PI-3-MEM -MEM suspension were used in theHA test: 1:10, 1:15, 1:20. 1:30, 1:40, 1:60, 1:80, 1:120, 1:160, 1:240,lz320. 1:480, 1:640, and 1:960. One ml. of the initial 1:10 and 1:15dilutions ofthe virus were prepared in Kahn tubes. and all succeeding HAand HI tests were performed using the above referred to microtiterprocedure.

One of the main features of an antigenantibody reaction is that thecombination is a firm and specific one. Therefore, if the antigen isparticulate, its corresponding homologous antibodies may easily beremoved from a mixture of antibodies. Since the reaction is specific,this facilitates determining the specific antigens or antibodies presentin the system, separating the antibodies if two or more kinds arepresent in the same serum, and concentrating the antibodies in theserum.

Tests were conducted to determine whether there was any immunologicalrelationship between the three viruses used in preparing the vaccinesdescribed above in order to be able to carry out quantitative studieswith the immune sera. It was necessary to adsorb out of the sera of theviral antibody components before the third component could be analyzed.The procedure was carried out in 13 X I00 mm.dilution tubes using immunesera against PI-3, IBR and BVD which had been prepared in rabbits. To a1.0 ml. aliquot of PI-3 immune serum diluted l:l0, was added 1.0 ml.containing a 10 TCID/ml. suspension of IBR virus. To this aliquot ofPI-3 immune serum diluted 1:10 was then added 1.0 ml. of 10 TCID/ml.suspension of BVD virus. The tubes were incubated at C. for 4% hours andwere titrated. Each of the other two remaining immune sera IBR and BVD,were treated in a similar manner by adsorbing out heterologousantibodies in the sera.

Comparative tests were made of adsorbed anc CATTLE COMPOSITION NUMBER OFAMOUNT IN- GROUP NO. OF VACCINES INJECTIONS JECTED, ML.

1 l-S NOT INJECTED 0 0 ll 6-8 ADJUVANT ONLY I 10 III 9-10 MINIMUMESSENTIAL MEDIUM (MEM) I I0 ONLY IV 11-15 ADJUVANT IBR 2 10 V 16-20ADJUVANT BVD 2 10 VI 21-25 ADJUVANT PI-3 2 10 VII 26-30 ADJUVANT IBR BVDPI-Il 10 VIII 31-36 IBR BVD PI-J W/O ADIUVANT I 10 IX 37-40 ADJUVANT IBRBVD PI-J 2 10 X 41-45 IBR BVD PI-3 W/O ADJUVANT 2 10 A microtitertechnique developed by Sever and described in Application of aMicrotechnique to Viral Serological Investigations," J. Immunol., 88(1962), pages 320-329, was used for the hemagglutinationactivity (HA)and hemagglutination-inhibition (HI) tests described hereinafter.

As to HA procedure, the myxovirus PI-3 (Strain Sf-4) is capable ofagglutinating the bovine, guinea pig, porcine and human type 0erythrocytes. The highest HA titer is reached when either guinea pig orporcine ml. saline solution). After thorough mixing, the samples wereleft at room temperature for 20 minutes and then centrifuged at 1500 rpmfor minutes. The supernatant fluid was saved. The serum samples werethen incubated at 56C. for 30 minutes to remove the complement. Thistreatment resulted in a serum dilution of 1:10. A 1:15 dilution of eachserum was obtained by adding 0.1 ml. of the 1:10 dilution to 0.5 ml.phosphate buffered saline the 1:10 and 1:15 diluted samples were used tomake the following dilutions in the HI test: 1:10, 1:15, 1:20, 1:30,1:40, 1:60, 1:80, 1:120, 1:160, 1:240, 1:320, 1:480, 1:640, and 1:960.

An HA test was performed on each stock virus suspension in MEM todetermine 4 HA units per 0.025 ml. After the dilution of the virus wasdetermined, a standard amount of4 HA units per sample was employed inthe H1 test.

0.025 ml. samples of a 1:10 dilution of serum and a 1:15 serum dilutionwere prepared. 0.025 ml. of phosphate buffered saline solution was addedto all samples. 0.025 ml. of the viral suspension (4 HA units per 0.025ml.) was then added to all samples and the samples were sealed. Afterincubation at room temperature (25C.) for 30 minutes, 0.05 ml. of 0.5percent guinea pig RBC was added to all samples. The samples wererescaled, incubated at room temperature for 30 minutes, and the resultsrecorded. Known negative and positive antisera were included in eachdetermination as controls. The H1 titer was established as the lastdilution in each group of samples to show complete. inhibition of HA.

A hermadsorption technique, using the bovine trachea cell linesupplemented the H1 test. When appropriate erythrocytes are added tocell cultures infected with influenza virus, the erythrocytes willadsorb to the infected cell surface. The phenomenon of hemadsorption isdependent upon selective attachment of erythrocytes onto the monolayersurface of tissue culture cells. It is demonstrated by the addition oferythrocytes to a tissue culture system in which propogation ofhemagglutinin-producing virus has occured.

Serum specimens were treated similarly to those in the H1 test. The serawere incubated at 56C. for 30 minutes to remove the complement and werethen diluted to 1:10 with phosphate buffered saline dilution tubes. A1:15 dilution of each serum was obtained by adding 0.1 ml. of the 1:10dilution to 0.05 ml. of phosphate buffered saline. The 1:10 and 1:15diluted samples were used to make the following dilutions used in thehemadsorption test: 1:10, 1:15, 1:120, 1:30, 1:40, 1:60, 1:80, 1:120,1:160, 1:240, 1:320 1:480, 1:640, and 1:960.

An HA test was performed on each stock virus suspension in MEM todetermine 4 HA units per 0.1 ml. After determining the dilution of virusthat contained 4 HA units, the dilution that contained 4 HA units couldbe calculated and used as the standard amount of virus for each bottlein the hemadsorption test.

The following procedure was used in carrying out the hemadsorptiontests. Bovine trachea cell monolayers were prepared in 1 02. bottleswith MEM plus fetal calf serum growth medium. The growth medium waspoured from the monolayers and the cell sheet was washed twice withphosphate buffered saline. Onetenth ml. of the appropriate virus-serummixture was added to each bottle, and the bottle was then rotated toassure complete coverage of the monolayer by the mixture. The monolayerswith the virus-serum mixtures were allowed to adsorb for 1 hour at roomtemperature (25C.). After the 1 hour adsorption period, 2.5 ml. of MEMmaintenance medium was added to each bottle, and the bottles were sealedand incubated at 37C. for 72 hours. The bottles were removed from theincubator and 0.4 ml. of a 0.5 percent suspension of washed guinea pigerythrocytes was added to each bottle, and the bottles were incubated at4C. for 30 minutes. Hemadsorption was observed in bottles where theunneutralized virus has infected the cells. Hemadsorption was notobserved in bottles where neutralized virus had failed to infect thecells. The hemadsorption titer was established as the highest dilutionto show a positive reaction.

Antibody with specificity for certain antigens of virus particles canneutralize the infectivity of the virus by combining with the viralantigen and preventing viral multiplication. Neutralizing antibody canbe detected by the inoculation of prepared virus antiserum mixtures intosusceptible animals or onto the surface of mammalian tissue cultures.The neutralization best demonstrates the appearance or rise in titer ofantibody during the course of illness or vaccination.

Tissue culture methods employing bovine trachea cells were utilized todetermine the neutralizing capacity of the produced antibody. Serialten-fold stock virus dilutions of l0" through l0"were prepared. Theantiserum was incubated at 56C. for 30 minutes to inactivate thecomplement and was substantially diluted to 1:10, 1:100 and 121000.Undiluted serum was also used. Non-immune serum was treated in the samemanner. One ml. of each virus dilution was combined with an equal amountof each antiserum dilution and incubated at room temperature for 1 hour.A 0.025 ml. quantity of each mixture was added to a suspended system ofbovine trachea cells. The bovine trachea cell concentration was 2.5 X10* cells per sample. Virusphosphate buffered saline mixtures andvirus-normal serum were used as controlsfThe virus-serum-cell system wasincubated for 6 days at 37C. at which time the cells were observed forcytopathological effects. The 1D, titers were determined by the Reed andMuench technique mentioned supra. and the neutralization indices (N1)were calculated bu subtracting the log of the titer of the virus mixedwith immune serum from the titer of the control virus.

In discussing the results of the above first study, the followingterminology will be used for convenience to designates the vaccinesemployed. The three monovalent vaccines are designated 1. Of these, 1Ais 1BR, 1B is BVD, and 1C is Pl-3. Vaccine ll refers to the polyvalentvaccine of the invention without the adjuvant and Vaccine 111 to thepolyvalent vaccine with adjuvant.

Cattle in Groups 11 through X, as shown in Table l, were injected with avaccine composed of either adjuvant only, MEM only, a single virus plusadjuvant (Vaccine l), triple virus without the adjuvant (Vaccine 11), ortriple virus plus adjuvant (Vaccine 111), and did not shown any evidenceof hypersensitivity or any other outward reactions as a result ofexperimental inoculations.

TABLE ll-continued AVERAGE ANTIBODY RESPONSE OF CATrLE INOCULATED Themeans values of the neutralizing activity of the antisera against IBRvirus are presented in Table II and FIG. I.

EITHER IBR VACCINE OR POLYVALENT VACCINE COMPOSED TABLE II OF PI-3, IBR,AND BVD VIRUSES, EXPRESSED AS NEUTRALIZATION INDICES AGAINST IBR VIRUSRECIPROCAL AVERAGE ANTIBODY RESPONSE OF CATTLE INOCULATED POST- OF ANTI-VACCINATION SERUM DIL.

VII VIII EITHER IBR VACCINE OR POLYVALENT VACCINE AGAINST IBR VIRUS VIIVIII RECIPROCAL POST OF ANTI- VACCINATION SERUM DIL.

ANIMALS IN GROUPS IV, IX. AND X WERE GIVEN A SECOND INJECTION OF ANTIGEN38 DAYS AFTER THE INTTIAL INJECTION. l5 ANTIBODIES NOT DETECTED INGROUPS I, II, AND III AT ANY TIME.

In FIG. I the neutralization index is plotted against the number of dayspost vaccination. The neutralizing antibodies to IBR virus were firstobserved in a l0 antiserum dilution on the 14th day after vaccination.Titrations of sera from earlier periods after vaccination antibodies.Consequently, these values are omitted from the tables. In animals fromGroup IV, injected twice with the Vaccine IA, neutralizing titersreached the maximum value by the 28th day after the second 35 injection.Similarly, in Group IX, the highest antibody response to two injectionsof Vaccine III also occurred at the same time. The neutralizationindices of the two groups, IV and IX, began decreasing at the 87th dayafte the initial vaccination. Sera from animals in Group VII, injectedonce with Vaccine III, reached maximum antibody activity on the 66th dayfollowing initial vaccination. Groups VIII and X which received one andtwo injections, respectively, of Vaccine II also reached a peak ofantibody activity at the 66th day after vacci- 66 nation. By the 87thday after vaccination, the neutralizing titers of the serum begandecreasing. Injection of vaccines without adjuvant did not produce ashigh an antibody response as did the vaccines that were prepared withthe adjuvant.

TABLE III AVERAGE ANTIBODY RESPONSE OF CATTLE INOCULATED WITH EITHER BVDVACCINE OR POLYVALENT VACCINE COMPOSED or PI-3. IBR, AND BVD VIRUSESEXPRESSED AS NEUTRALIZATION INDICES AGAINST BVD VIRUS SERUM OBTAINEDRECIPROCAL ON INDICATED OF ANTI- DAY POST-VACCINATIOISERUM DIL. V

VIII

VII

a8 Asbeoas i hssj 0 02 02 03 0032 02 002 002 O I0 I00 I000 I0 I00 I000TABLE Ill-continued SERUM OBTAINED RECIPROCAL ON INDICATED OF ANTI- DAYPOST-VACCINATIOISERUM DIL. V

ANIMALS IN GROUPS V. IX. AND X WERE GIVEN A SECOND INJECTION OF ANTIGENON 38 DAYS AFTER THE INITIAL INJECTION. NO ANTIBODIES DETECTED IN GROUPSI. II. AND III AT ANY TIME.

As in the case of IBR, antibodies to BVD were first 20 observed in thel0 antiserum dilution at the l4th day vaccination. Two injections ofvaccine IB caused an antibody response that reached a maximum level onthe 28th day following injection. By the 87th day fol- 2 lowing theinitial vaccination, the neutralizing activity of the immune serum beganto decrease. The animals in Group VII, which received one injection ofVaccine III. produced a maximum response on the 66th day afterinjection, but the antibody activity of the sera decreased by the 87thday. The cattle in Group IX, receiving two injections of Vaccine III,reache a maximum level of antibody production at the l4th day subsequentto the second injection and began decreasing by the 49th day after thesecond inoculation. Animals 3 in Group VIII, having received a singleinjection of Vaccine ll, reached their peak of neutralizing activityagainst BVD at the 45th day after vaccination. and the antibody ltiterof the immune serum began decreasing by the 52nd day post vaccination.Cattle in Group X. injected twice with Vaccine II, reached a peak ofneutralizing activity at the 28th day after the second injection, andthe antibody titer began decreasing by the 49th day subsequent to thesecond injection.

The HI activity of the immune sera against PI-3 virus HI antibodiestoward PI-3 were first observed on the 14th day after vaccination.Maximum HI activity of the sera from cattle in Group VI, having receivedtwo injections of the Vaccine IC, was observed at the 28th day followingthe second injection, and the titer began decreasing by the 49th dayafter the second inoculation. Sera from Group VII, which recieved oneinjection of Vaccine III, reached a maximum HI titer at 66 days aftervaccination. By the 87th day following vaccination, the HI activity ofthe immune sera began to decrease. Cattle in Group IX, which receivedtwo injections of Vaccine III, also produced a maximum antibodyresponses to PI-3 at the 66th day following the initial injection. Theantibody level of the sera decreased by the 87th day after the initialvaccination. Animals in Group X, inoculated twice with the Vaccine II,produced a maximum response to PI-3 28 days following the secondvaccination, and by the 49th day subsequent to the second injection theantibody level began to decrease.

Antibody activity of the sera toward PI-3 was also determined by theprocedure of hemadsorption inhibition (HAdI). The mean values of theHAdI activity are recorded in Table V and FIG. '4.

TABLE V is presented in Table IV and FIG. .3.

TABLE IV AVERAGE ANTIBODY RESPONSE OF CATTLE INOCULATED EITHER PI-3VACCIIEIIE OIZJEIbYVALENT VACCINE OF PI-3. IBR. AND BVD VIRUSESEXPRESSED AS RECIPROCAL OF HI ACTIVITY AGAINST Pl-3 SERUM OBTAINED GROUPON INDICATED DAY POST-VACCINATION AVERAGE ANTIBODY RESPONSE OF CATTLEINOCULATED WITH EITHER PI-3 VACCINE OR POLYVALENT VACCINE COMPOSED OFPl-3. IBR, AND BVD VIRUSES EXPRESSED AS RECIPROCAL OF HEMADSORPTIONACTIVITY AGAINST PI-3 SERUM OBTAINED ON INDICATED DAY VI VII VIII IX XPOST-VACCINATION VI VII VIII IX X 3 10 I0 I0 I0 10 3 10 I0 10 I0 10 I010 I0 10 I0 I0 I0 I0 I0 I0 I0 l0 I4 22 I0 I6 10 IS I4 26 I2 I8 I0 I7 I738 20 30 I8 26 I7 42 I8 30 I9 28 23 72 30 34 34 34 23 76 34 36 38 38 3|I00 64 64 52 48 31 I04 72 68 60 56 35 220 I04 72 I08 35 25 30 44 30 38U. '0 i fit it B 45 335 I92 I04 272 I20 45 352 203 04 233 20 52 448 288I28 384 I76 52 448 288 I36 4I6 I92 66 480 352 I44 416 208 66 SIZ 384 I44480 208 87 384 304 I28 368 I I2 87 416 336 I20 384 I I2 I20 288 256 92272 84 6 I20 288 272 84 272 88 ALL TITERS OF I0 ARE I0. THE INDICATED HITITERS ARE BASED ON A RISE ABOVE A BASE LINE OF I0. GROUPS I. II. ANDIII SHOWED NO RISE IN ANTIBODY TITER ABOVE A BASE LINE OF I0.

" SECOND INJECTION.

-NOT DONE.

ALL TITERS OF I0 ARE l0. THE INDICATED HEMADSORPTION TITERS ARE BASED ONA RISE ABOVE A BASE LINE OF I0. GROUPS I. II, III SHOWED NO RISE INANTIBODY TITER ABOVE A BASE LINE OF I0.

" SECOND INJECTION.

-NOT DONE.

13 The Mill titers parallel the III titers. but the llAdl test is moresensitive than the HI test. An analysis of the HAdl activity of theimmune sera toward Pl-3 would be repetitious of the HI activityanalysis. An inspection of the HAdI titers in Table V and FIG. 4 revealsthe similarity in antibody activity to HI titers found in Table IV andFIG. 3.

The average neutralizing activity of the immune sera toward PI-3 isrecorded in Table VI and FIG. 5

TABLE VI I4 tion. and by the 49th day alter the second inoculationAVERAGE ANTIBODY RESPONSE OF CATTLE INOCULATED WITH EITHER Pl-3 VACCINEOR POLYVALENT VACCINE COMPOSED OF PI-3, IBR, AND BVD VIRUSES EXPRESSEDAS NEUTRALIZATION INDICES AGAINST PI-J VIRUS DAY RECIPROCAL POST- OFANTI- VACCINATION SERUM DIL.

I0 I00 I000 0 I0 I00 I000 0 I0 I00 I000 0 I0 I00 I000 0 seq this ANIMALSIN GROUPS VII IX. AND X WERE GIVEN A SECOND INJECTION OF ANTIGEN IIIIDAYS AFTER THE INITIAL INJECTION. ANTIBODIES NOT DETECTED IN GROUPS I.II, AND III AT ANY TIME.

Antibodies toward Pl-3 were first demonstrated in a higher level ofantibodies than did the complex vaccin l0 dilution of antiserum 14 daysafter initial vaccination. Maximum neutralizing activity of the serum inthe cattle of Group VI, which received two injections of the Vaccine IC,was recorded on the 28th day after the second injection. The antibodylevel began decreasing by the 49th day subsequent to the secondinoculation. Animals in Group VII, injected once with Vaccine III,developed a maximum response to PI-3 66 days following the initialvaccination. By the 87th day post vaccination, the NI of neutralizationindex began to decrease. Maximum neutralizing activity of sera fromGroup VIII, having received one injection of Vaccine II, was reached atthe 66th day after the initial injection and began decreasing by the87th day post vaccination. Animals in Group IX. which received twoinjections of Vaccine III, developed maximum neutralization titers inthe sera at the 28th day following the second injecwithout the adjuvant(Vaccine II). Antibodies to th three viruses persisted for over 4 monthsin the sera c animals injected twice with the adjuvant-containinvaccine. The test results also indicate that the thre killed viruses ofthe polyvalent vaccines of the inver tion do not interfere with eachother and that the v ruses may be administered in a single polyvalentvar cine.

A second study was carried out employing three vac cines. The firstvaccine was a polyvalent vaccine (Vac cine I) consisting of equalproportions of bovine virz diarrhea virus, Southern Illinois Universitystrai (BVD-SIU). infectious bovine rhinotracheitis viru: SouthernIllinois University strain (lBR-SIU) and p2 rainfluenza-3 (SF-4),Southern Illinois University strai (Pl-3-SIU). The second vaccine(Vaccine II) was polyvalent vaccine consisting of equal proportions cthe above three viruses and of Pasteurella multocida, an organismfrequently isolated along with PI-3 in cases of shipping fever. Thethird vaccine (Vaccine III) was a polyvalent vaccine consisting of equalproportions of BVD and [ER viruses (Goff strains obtained fromAffiliated Laboratories, Whitehall, Illinois) and the Pl-3- SIU virus.Each of the vaccines was prepared with a sodium alginate adjuvantcomposed of an aqueous solution containing 4.0 percent weight/volume ofsodium alginate and 0.67 percent by weight calcium gluconate. Theadjuvant was mixed in a 1:1 ratio with the suspension of viruses for thecomplete vaccine in each instance.

The Pl-3-SIU virus employed was serially passed 5 times in bovineembryonic trachea (BEmT) cells and a hemagglutination (HA) titer of1:640 was achieved. The lBR-SIU and lBR-Goff viruses were seriallypassed 6 times each and tissue culture infective dose (TCID titers of10" and 10 respectively, were reached. The BVD-SIU and BVD-Goff viruseswere serially passed seven times in BEmT cells and TCID titers of 10"and 10, respectively, were achieved. Vacine 111 which incorporatedPasteurella multocida contained 1 X 10 organisms per ml.

All viruses employed were propagated as previously described and eachvirus was inactivated with 0.4 percent formalin (final concentration)and maintained for 1 week at 4C. All other procedures employed in thefirst study were followed in the second study.

Thirty-six head of beef cattle, 22 heifers and 14 steers, of Herefordbreed, each weighing 750-950 pounds, were employed as test animals inthe second study. The animals were 15 months old. They had no history ofvaccination and there had been no history of clinical cases of shippingfever, 1BR, or BVD in the herd.

The 36 head of cattle were divided into five groups. The vaccines wereparenterally administered by inoculating the animals intramuscularly inthe biceps femoris 16 TABLE V111 AVERAGE ANTIBODY RESPONSE OF CATTLEINOCULATED WITH POLYVALENT VACCINE COMPOSED OF P1-3, 1BR. AND

BVD VIRUSES EXPRESSED AS NEUTRALIZATION INDICES AT A 1:100 ANTISERUMDILUTION AGAINST lBR-SIU 2nd lnjectlon of original vacclne Figures buedon 1 H000 lltllltfllm dilution Neutralizing antibody was firstdiscovered in a 1:100 dilution of antiserum in the animals in Groups 11,Ill, and IV, 14 days following the initial vaccination. The titerscontinued to rise, and when the neutralization index of 1.0 was reached,the second injection of the same vaccine was administered. Groups II andIV received the second inoculation on the 51st day and Group III on the37th day following the initial injection. The maximum neutralizingtiters in Group II were achieved on the 14th day following the secondinjection or on the 65th day after the initial injection. Maximumneutralizing activity of sera from Group 111 was seen on the 28th dayfollowing the second injection.

Antibody activity to lBR-SIU was still present in the sera from Groups11, Ill, and IV 93 days following the first injection.

Table IX and FIG. 7 show the neutralizing activity of antisera ofanimals in Groups 11, III, and 1V against BVD-SIU.

TABLE 1X AVERAGE ANTIBODY RESPONSE OF CATTLE INOCULATED WITH POLYVALENTVACCINE COMPOSED OF PI-3. 1BR, AND BVD VIRUSES EXPRESSED ASNEUTRALIZATION INDICES AT A 1:100 ANTISERUM DILUTION AGAINST IBR-SIUmuscle using disposable l9-gauge needles and syringes. g GROUPS The Sameprocedures described above for the first VACCINATION l n m w study wereused in carrying out the same tests In the I 00 0 00 00 second study. 300 00 0.0 Table V11 shows the composition of the vaccines and Z 8-8 8g88 8:? the injection protocol employed in the second study. 4 0.7 m

TABLE V11 COMPOSITION OF VACClNES AND INJECTION PROTOCOL FOR CATTLE 1NGROUPS l-V COMPOSrrION NUMBER OF AMOUNT IN- GROUP CATTLE NO. OF VACCINESINJECTIONS .IECTED. ML.

1 7 Not injected 0 0 ll 7 SIU strains of 1BR. BVD, PI-3 2 5 111 7 SIUstrains of 1BR. BVD. Pl-3 2 10 1V 7 SIU strains of 1BR. BVD. PI-3 andPast. multocida 2 5 v a lBR-Goff, BVD- Goff, Pl-3-S1U 2 5 The proceduresfor determining the antibody levels 8 8 I g 3.; in the sera of animalsin the second study were slmllar 5 J to that of the first study, exceptfor two minor varIa- 65 on M 33 3.0 tions which did not affect theresults. 79 0.0 3.4 3.0 2.21 The means neutralization values of antiseraagaInst 93 0.0 3.1 "2.8 2.4

lBR-SIU virus are presented in Table V111 and FIG. 6.

2nd injection of original vaccine "Figures based on a 1:1(100 antiserumdilution As with IBR-SIU, neutralizing antibody was first detected onthe 14th day after the initial injection. In Groups II and IV, themaximum titers were observed on the l4th day following the secondinjection. Maximum neutralization activity in Group III was seen on the28th day after the second inoculation. Antibody to IBR-SIU persisted inthe sera 93 days after the initial injection.

The hemagglutination-inhibiting activity of the sera against PI-3 virusis seen in Table X and FIG. 8.

TABLE X AVERAGE ANTIBODY RESPONSE OF CATTLE INOCULATED WITH POLYVALENTVACCINE COMPOSED OF PI-Il, IBR.

All titers of IO i II). The indicated HI liters are based on a riseabove a base line of IO.

Parainfluenza-Zl-SIU is the only virus common to all vaccines of GroupsII-V. Sera from Groups II, IV, and

TABLE xn AVERAGE ANTIBODY RESPONSE OF CATI'LE INOCULATED WITH POLYVALENTVACCINE COMPOSED OF PI-3. IBR-GOFF. AND BVD-GOFF VIRUSES EXPRESSED ASNEUTRALIZATION INDICES AT A l:l00 ANTISERUM DILUTION AGAINST BVD-GOFFDAY GROUPS POST-VACCINATION I V I I 0.0 0.0 14 0.0 0.I 2I 0.0 0.9 37 0.01.] SI 0.0 2.4 65 0.0 3.5 79 0.0 3.1 93 0.0 2.8

2nd injection of original vaccine Neutralizing antibody was firstdetected on the 14th day following the initial injection. Maximum titerswere achieved on the 65th day after the first injection or on the I4thday following the second injection. The presence of antibody was stilldetected on the 93rd day after the first vaccination.

The agglutinating activity of the antisera ofGroup IV againstPasteurella mulmcida is shown in Table XIII.

TABLE XIII AVERAGE ANTIBODY RESPONSE OF CATTLE INOCULATED WITHPOLYVALENT VACCINE COMPOSED OF I'l-3 IBR. DVD, AND PAST. MULTOCIDAEXPRESSED AS RECIPROCAL OF AGGLUTINATING ACTIVITY AGAINST PAST.MULTOCIDA DAY GROUPS V showed maximum HI titers on the 14th dayfollowing POSTNACCINATION l w the second inoculation. The maximum HItiter in I 10 10 Group III was on the 28th day after the second inocula-3 10 I0 tion. Hemagglutination-inhibition titers persisted in the Z :8:g sera after the 93rd post-vaccination day. 5 :t :8 Table XI and FIG. 9show the neutralizing activity of 37 lo 0 .51 10 '10 antisera againstIBR Goff VII'LIS. 65 '0 lo 79 I0 10 Table XI 93 10 I0 AVERAGE ANTIBODYRESPONSE OF CATTLE INOCULATED WIT POLYVALENT VACCINE COMPOSED OF PI-3.IBR-GOFF. AND BVD-GOFF VIRUSES EXPRESSED AS NEUTRALIZATION INDICES AT Al:l00 ANTISERUM DILUTION AGAINST lBR-GOFF DAY GROUPS POST-VACCINATION 70.0 0.0 l l 0.0 00 I4 0.0 0.4 2i 0.0 L0 37 0.0 1.8 Sl 0.0 '3.I 65 0.04.3 79 0.0 4.I 93 0.0 3.7

2nd injection of original vaccine Maximum neutralizing activity wasachieved on the 14th day following the second injection. Titers werefirst detected on the 14th day following the initial injection.Neutralizing activity was still present on the 93rd day following theinitial injection.

The mean neutralizing activity of sera to BVD-Goff is shown in Table XIIand FIG. 9.

2nd injection of original vaccine All titen of I0 & l0. The indicatedagglutinatint titers are based on a rise above 1 base line of IO.

No agglutinating titers were detected in the sera ol animals injectedwith Vaccine II. The inability of ani' mals to develop detectableantibody toward Past. multocida is unexplainable. Even after a secondinjection 01 antigen, agglutinating titers were not achieved.

The results of these two studies show that high titer: of antibodies toBVD, IBR, and PI-3 viruses were detected in animals inoculated with thekilled polyvalen' vaccine of the invention and that these titers werecon siderably higher when the polyvalent vaccine wa: mixed with thealginate adjuvant. In addition, the titer: of antibodies to each virusobtained after injection 0 the polyvalent vaccines were of the sameorder of mag nitude as those produced by inoculation of single virupreparation.

In view of the above, it will be seen that the severe objects of theinvention are achieved and other advan tageous results attained.

As various changes could be made in the above methods and productswithout departing from the scope of the invention. it is intended thatall matter contained in the above description or shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

What is claimed is:

l. A vaccine comprising a suspension of l to percent weight/volume of asoluble alginate adjuvant vehicle suitable for parenteral injection andan admixed volume of an aqueous solution containing a finalconcentration of between about 0.3percent and 0.5 percent formaldehydeand formalin-killed bovine viral diarrhea virus in a concentration oflO-l0 TClD /ml. which has been maintained at a temperature of about 4C.for approximately 1 week.

2. A vaccine as set forth in claim 1 wherein said vehicle is constitutedby an aqueous 0.4 percent solution of formaldehyde.

3. A vaccine as set forth in claim I wherein said adjuvant isconstituted by an equal volume of an aqueous solution containing about Ito 5 percent weight/volume of a soluble alginate having the followingcharacteristics:

a. in 5 percent weight/volume aqueous solution.

readily passes a 24-gauge needle;

b. in 4 percent weight/volume aqueous solution, has

a viscosity less than 50 centistokes; c. a chemical equivalence below250; d. a milliosmolarity less than kg. of water on a Fiske osmometer.

# t i i i

1. A VACCINE COMPRISING A SUSPENSION OF 1 TO 5 PERCENT WEIGHT/VOLUME OFA SOLUBLE ALGINATE ADJUVANT VEHICLE SUITABLE FOR PARENTERAL INJECTIONAND AN ADMIXED VOLUME OF AN AQUEOUS SOLUTION CONTAINING A FINALCONCENTRATION OF BETWEEN ABOUT 0.3 PERCENT AND 0.5 PERCENT FORMALDEHYDEAND FORMALIN-KILLED BOVINE VIRAL DIARRHA VIRUS IN A CONCENTRATION OF10*:5-10**8 TCID50/ML. WHICH HAS BEEN MAINTAINED AT A TEMPERATURE OFABOUT 4*C. FOR APPROXIMATELY 1 WEEK.
 2. A vaccine as set forth in claim1 wherein said vehicle is constituted by an aqueous 0.4 percent solutionof formaldehyde.
 3. A vaccine as set forth in claim 1 wherein saidadjuvant is constituted by an equal volume of an aqueous solutioncontaining about 1 to 5 percent weight/volume of a soluble alginatehaving the following characteristics: a. in 5 percent weight/volumeaqueous solution, readily passes a 24-gauge needle; b. in 4 percentweight/volume aqueous solution, has a viscosity less than 50centistokes; c. a chemical equivalence below 250; d. a milliosmolarityless than 150 kg. of water on a Fiske osmometer.